Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity sequence domains (LCDs), but how these LCDs drive transactivation remains unclear. We used live-cell single-molecule imaging to reveal that TF LCDs form local high-concentration interaction hubs at synthetic and endogenous genomic loci. TF LCD hubs stabilize DNA binding, recruit RNA polymerase II (RNA Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. Under physiological conditions, rapid and reversible LCD-LCD interactions occur between TFs and the RNA Pol II machinery without detectable phase separation. Our findings reveal fundamental mechanisms underpinning transcriptional control and suggest a framework for developing single-molecule imaging screens for drugs targeting gene regulatory interactions implicated in disease.

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation, with the shorter yeast CTD forming less-stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association, whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters or hubs at active genes through interactions between CTDs and with activators and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.

Animal genomes are folded into loops and topologically associating domains (TADs) by CTCF and loop extruding cohesins, but the live dynamics of loop formation and stability remain unknown. Here, we directly visualize chromatin looping at the Fbn2 TAD in mouse embryonic stem cells using super-resolution live-cell imaging and quantify looping dynamics by Bayesian inference. Unexpectedly, the Fbn2 loop is both rare and dynamic, with a looped fraction of ~3-6.5% and a median loop lifetime of ~10-30 min. Our results establish that the Fbn2 TAD is highly dynamic, where ~92% of the time cohesin-extruded loops exist within the TAD without bridging both CTCF boundaries. This suggests that single CTCF boundaries rather than the fully CTCF-CTCF looped state may be the primary regulators of functional interactions.

Sequence-specific DNA-binding activators, key regulators of gene expression, stimulate transcription in part by targeting the core promoter recognition TFIID complex and aiding in its recruitment to promoter DNA. Although it has been established that activators can interact with multiple components of TFIID, it is unknown whether common or distinct surfaces within TFIID are targeted by activators and what changes if any in the structure of TFIID may occur upon binding activators. As a first step toward structurally dissecting activator/TFIID interactions, we determined the three-dimensional structures of TFIID bound to three distinct activators (i.e., the tumor suppressor p53 protein, glutamine-rich Sp1 and the oncoprotein c-Jun) and compared their structures as determined by electron microscopy and single-particle reconstruction. By a combination of EM and biochemical mapping analysis, our results uncover distinct contact regions within TFIID bound by each activator. Unlike the coactivator CRSP/Mediator complex that undergoes drastic and global structural changes upon activator binding, instead, a rather confined set of local conserved structural changes were observed when each activator binds holo-TFIID. These results suggest that activator contact may induce unique structural features of TFIID, thus providing nanoscale information on activator-dependent TFIID assembly and transcription initiation.[Keywords: TAF; TFIID; transcription; activator; structure] Supplemental material is available at http://www.genesdev.org.

authors note errors in three sentences in the second paragraph on page 15042, left column. The corrected paragraph is reprinted below, and the revised sentences appear in boldface. The authors are grateful to Dr. Benny Abraham for identifying the errors.The reported SNP for CD24 is a replacement of C at nucleotide 226 by T (C3T) in the coding region of exon 2 (GenBank accession no. NM013230), which results in a substitution of Ala at amino acid 57 by Val near the GPI anchorage site of the mature protein. The genomic DNA was isolated from Ϸ5 ϫ 10 6 human peripheral blood leukocytes (PBL) by using the QIAamp DNA Blood Minikit (Qiagen, Valencia, CA). DNA fragments bearing this SNP site were amplified by PCR by using a forward primer (TTG TTG CCA CTT GGC ATT TTT GAG GC) and a reverse primer (GGA TTG GGT TTA GAA GAT GGG GAA A). The PCR conditions were as follows: 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min, for 35 cycles. The predicted CD24 PCR fragment is 453 bp long. The C3T change yielded a BstXI restriction enzyme site at nucleotide 225, which allowed us to differentiate these two different CD24 alleles by restriction fragment length polymorphism analysis. Briefly, an aliquot of CD24 PCR products was digested with BstXI for 16 h at 50°C. The digested products were then separated in a 2.5% agarose gel. The predicted digestion pattern is as follows: PCR products of T226 allele will be cut into two small fragments (325 and 129 bp), whereas those of the C226 will be completely resistant. A combination of the two types of the products at close to 50% levels indicates the heterozygosity of the subject. 1073͞pnas.0501422102), the authors note the following regarding the homology of conceptual translations of putative noncoding RNA (pncr) transcripts to known proteins. The report correctly states that for all candidate noncoding transcripts curated in this study, BLASTX analyses using default parameters return no results. However, subsequent analyses of those candidates designated by this study as pncr genes using BLASTP with a PAM30 substitution matrix has revealed homology to known proteins for 2 of the 17 genes listed in Table 2: pncr005:2R and pncr006:X. Homology to a conceptual translation was found for a third transcript, pncr007:3R. We are therefore withdrawing the pncr gene designations in these three cases. No protein homology is detected for other pncr transcripts under these parameters.

Achieving a quantitative and predictive understanding of 3D genome architecture remains a major challenge, as it requires quantitative measurements of the key proteins involved. Here, we report the quantification of CTCF and cohesin, two causal regulators of topologically associating domains (TADs) in mammalian cells. Extending our previous imaging studies (Hansen et al., 2017), we estimate bounds on the density of putatively DNA loop-extruding cohesin complexes and CTCF binding site occupancy. Furthermore, co-immunoprecipitation studies of an endogenously tagged subunit (Rad21) suggest the presence of cohesin dimers and/or oligomers. Finally, based on our cell lines with accurately measured protein abundances, we report a method to conveniently determine the number of molecules of any Halo-tagged protein in the cell. We anticipate that our results and the established tool for measuring cellular protein abundances will advance a more quantitative understanding of 3D genome organization, and facilitate protein quantification, key to comprehend diverse biological processes.

Achieving a quantitative and predictive understanding of 3D genome architecture remains a major challenge, as it requires quantitative measurements of the key proteins involved. Here we report the quantification of CTCF and cohesin, two causal regulators of topologically associating domains (TADs) in mammalian cells. Extending our previous imaging studies (Hansen et al., 2017), we estimate bounds on the density of putatively DNA loop-extruding cohesin complexes and CTCF binding site occupancy. Furthermore, co-immunoprecipitation studies of an endogenously tagged subunit (Rad21) suggest the presence of cohesin dimers and/or oligomers. Finally, based on our cell lines with accurately measured protein abundances, we report a method to conveniently determine the number of molecules of any Halo-tagged protein in the cell. We anticipate that our results and the established tool for measuring cellular protein abundances will advance a more quantitative understanding of 3D genome organization, and facilitate protein quantification, key to comprehend diverse biological processes. RESULTS Determining the number of CTCF and cohesin proteins per cell

Matrix metalloproteinases (MMPs) are important for developmental tissue remodeling and for the inflammatory response. Although the vertebrate MMP family is large and functionally redundant, the fruitfly Drosophila melanogaster has only two MMPs, both essential genes. Our previous work demonstrated that Mmp1 is required for growth of the tracheal system, and we suggested that the mutant phenotype resulted from aberrant persistence of cell adhesion to the extracellular matrix. Here we report the identification of NijA, a transmembrane protein whose vertebrate homologs regulate cell adhesion, as a two-hybrid binding partner for Mmp1. The binding of Mmp1 and NijA was confirmed by coimmunoprecipitation of endogenous proteins from flies, and the endogenous proteins were found to colocalize at the tracheal cell surface in larvae. When NijA is expressed in S2 cells, they lose adhesion to surfaces; this adhesion-loss phenotype is dependent on the expression and catalytic activity of Mmp1. Our data indicate that Mmp1 releases the N-terminal extracellular domain of NijA. This liberated ectodomain promotes the loss of cell adhesion in a cell-nonautonomous manner. We suggest that tracheal cell adhesion is regulated by a novel mechanism utilizing an MMP and a ninjurin family member.